Biomimetic Generation and Remodeling of Phospholipid Membranes

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Biomimetic Generation and Remodeling of Phospholipid Membranes by Dynamic Imine Chemistry Andres Seoane Fernandez, Roberto J. Brea, Alberto Fuertes, Kira Podolsky, and Neal K Devaraj J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018

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Journal of the American Chemical Society

Biomimetic Generation and Remodeling of Phospholipid Membranes by Dynamic Imine Chemistry Andrés Seoane, Roberto J. Brea, Alberto Fuertes, Kira A. Podolsky, and Neal K. Devaraj* Department of Chemistry and Biochemistry University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093, United States

Supporting Information Placeholder ABSTRACT: Biomimetic liposomes have a wide array of applications in several areas, ranging from medicinal chemistry to synthetic biology. Due to their biocompatibility and biological relevance, there is particular interest in the formation of synthetic phospholipid vesicles and the development of methods to tune their properties in a controlled manner. However, while true biological membranes are capable of responding to environmental stimuli by enzymatically remodeling their composition, synthetic liposomes are typically static once formed. Herein we report the chemoselective reaction of the natural amine-containing lysosphingomyelin with a series of longchain aldehydes to form imines. This transformation results in the formation of phospholipid liposomes that are in dynamic equilibrium with the aldehyde-amine form. The reversibility of the imine linkage is exploited in the synthesis of vesicles that are capable of responding to external stimuli such as temperature or the addition of small molecules.

Phospholipid membranes are essential features of every living organism, as they define boundaries and provide needed compartmentalization for executing metabolic reactions.1 The synthesis of phospholipids in cells is carried out by membrane-bound acyltransferases which link a single-chain lysophospholipid and an activated fatty acid.2 In addition to their ability to synthesize phospholipids from single-chain precursors, organisms can tune the properties of their lipid membranes by replacing the aliphatic chain of their components through the action of phospholipases, transacylases, and acyltransferases (Figure 1A).3 Inspired by these transformations, several nonenzymatic systems have been created, allowing the de novo formation of artificial phospholipid membranes.4-7 Remodeling of in situ generated membranes can be achieved by leveraging the ability of Nmethylated cysteine-modified lysolipids to undergo reversible native chemical ligation (RNCL) with thioesters. This process resembles the activity of natural transacylases and is able to replace both headgroups and acyl tails in an efficient manner.8 Despite being a step toward the facile remodeling of synthetic membranes, there are several drawbacks to this technique. First, since the method is based on transacylase activity, the addition of an entirely new lysolipid is required to replace one of the aliphatic tails, leading to byproducts of high complexity and size. In addition, the ratio of the starting versus remodeled lipids in the equilibrium can only be controlled by adding a high excess of one of the lysolipids. This feature is due to the similar stability of the different amides formed in the process. Therefore,

a system able to replace only the acyl chains of the phospholipid, mimicking phospholipase/acyltransferase activity, would be a significant advance in the biomimicry of membranes. Furthermore, the ability to drive the equilibria based on the stability of the different phospholipids would enable applying the principles of dynamic combinatorial chemistry (DCC) to phospholipid membranes.

Figure 1. Construction of lipid membranes. A) Natural pathways for de novo phospholipid synthesis and remodeling [PL: phospholipase, AT: acyltransferase, TA: transacylase]. B) Biomimetic assembly of membrane-forming lipids by a reversible imine linkage represented by the reaction between the lysosphingomyelin 1 and the (Z)-9octadecenal (2a) to afford sphingolipid 3a, which self-assembles in situ to form micron-sized vesicles. To meet these demands, we required a reversible transformation whose product stability could be readily tuned. One of the most important reversible reactions in organic chemistry is the formation

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of imines from amines and aldehydes, leading to a dynamic system that can be tuned by the careful choice of both reaction partners, as well as other parameters such as temperature or pH.9,10 Reversible imine formation is a cornerstone of several fields including organocatalysis,11,12 supramolecular chemistry13,14 and DCC.15-16 Despite the potential of this reaction to generate robust and biocompatible membranes under physiological conditions, there are limited examples of imine-based vesicles. Moreover, their composing amphiphiles lack resemblance of natural phospholipids.17,18 Herein we demonstrate that a natural amine-containing lysophospholipid, lysosphingomyelin, reacts chemoselectively with long-chain aldehydes to form imines, driving the de novo generation of dynamic biomimetic phospholipid liposomes (Figure 1B). We also show how the reversibility of the imine can be used to engineer vesicles capable of responding to external stimuli. Initially, we combined lysosphingomyelin (1) and the long-chain aliphatic oleylaldehyde (2a), whose reaction would give rise to 3a, a mimic of the naturally occurring N-oleoyl-D-erythrosphingosylphosphorylcholine (Figure 1B).19 Although the equilibrium of imine formation in water is usually shifted towards the starting materials,20 it has been shown that the formation of amphiphiles capable of self-assembling can lead to the stabilization of the imine, presumably by shielding the electrophilic carbon of the imine from water molecules.21-25 The reaction between 1 and 2a was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) aqueous buffer at pH 7.5 under millimolar concentrations. Immediately after adding both reaction partners to the buffer, we observed the presence of oil droplets by microscopy. Ten minutes after the mixing, tubules and vesicles began to appear on the surface of the droplets (Figure 2A) until the oil droplets were fully consumed and only liposomes remained in solution (Figure 2B, Figure S3C). The formed vesicles were characterized by transmission electron microscopy (TEM) (Figure 2C, Figure S4). When the resulting mixture was analyzed by LC-MS and evaporative light-scattering detection (ELSD), negligible quantities of the proposed product were detected (Figure S5A). We reasoned this was likely due to hydrolysis of the imine during the elution. To avoid such decomposition and to reproducibly capture the different species in the solution, the imine equilibrium was frozen by the addition of an excess of NaBH4,18 which reduced the proposed iminophospholipid 3a to the corresponding stable aminophospholipid 4a (Figure 2D). Remarkably, the imine formation exhibits a high chemoselectivity towards the lysosphingomyelin. When the reaction was carried out in the presence of other biologically relevant amines (adenine, glucosamine and lysine), the main product detected was 4a (Figure S1). This effect is most likely due to the hydrophobic effect, which leads to the retention of 2a in the bilayer favoring the reaction of 1 over the other amines.26 We next explored the ability of other aldehydes to form imines and vesicles upon reaction with lysosphingomyelin 1 (Figure 3). Adding short-chain aldehydes (2b,c) to 1 did not result in the formation of liposomes and, after reduction of the mixtures, no amines were detected (Figure 3C). This behavior can be explained by the inability of the putative imines 3b,c to assemble into bilayers (Figure S3). Without the formation of liposomes, the imines would lack the necessary protection from hydrolysis. Interestingly, aromatic long-chained aldehydes such as 4-n-dodecyloxybenzaldehyde (2d) also led to the formation of liposomes (Figure S3D). To obtain hydrolysis-resistant iminophospholipids, 4-n-octyloxysalicylaldehyde (2e), which provides extra stabilization through intramolecular hydrogen bonding

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between the imine and ortho hydroxy group,27,28 was added to the lysosphingomyelin 1. Combining 1 and 2e resulted in the formation of vesicles under the standard reaction conditions (Figure S3E). Moreover, the equilibrium is completely shifted to the imine (Figure 3C). Remarkably, this imine can be detected by LC-MS without the need for reduction (Figure S5E) proving the ability of hydrogen bonding to significantly stabilize imine formation in water, even at slightly acidic pH.

Figure 2. Generation of vesicles driven by imine formation. Fluorescence microscopy images of a mixture of 1 (1 mM), 2a (1 mM) and Nile red (1 µM) in HEPES buffer pH 7.5 after 10 min (A) and 14 h (B). Scale bars denote 5 µm. C) TEM images of the vesicles composed of 3a. Scale bar denotes 250 nm. D) HPLC/ELSD traces of the reaction between 1 and 2a after reduction with NaBH4, showing the formation of amine-containing sphingolipid 4a. Having demonstrated the generation of phospholipid vesicles by imine formation from various aldehydes, we next studied how the dynamic covalent system would respond to external stimuli (Figure 3D). A classic phenomenon observed in living organisms is the ability of cells to change their membrane composition in response to external temperature.29 Intriguingly, we found that a solution of two equivalents of oleylaldehyde 2a and one equivalent of benzyl aldehyde 2d with lysosphingomyelin 1 responded to temperature changes by shifting the composition of the species in the equilibrium (Figure 3D). When the reaction was carried out at 37 ˚C for 16 h, the major product obtained after reduction was the benzylamine 4d. Lowering the temperature resulted in a higher proportion of oleyl-based aminophospholipid 4a, which became the main component of the mixture when stirred at 23 ˚C (Figure 3D). This response to external conditions can be explained by kinetic versus thermodynamic control.30 At low temperature, when hydrolysis is slow, the product is controlled by the rate of formation of imines leading to oleylimine 3a, due the higher electrophilicity of aldehyde 2a.31 However, at increased tem-

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Journal of the American Chemical Society

peratures, hydrolysis has to be taken in account, favoring the formation of the more thermodynamically stable conjugated aromatic imine.32

blance to the Land’s cycle of membrane remodeling in cells, where one acyl chain is removed and a new one reattached enzymatically.3 The successful remodeling of the iminophospholipids prompted us to investigate potential applications. We found that the addition of a salicylaldehyde without an aliphatic substituent (2f) to 3a also resulted in imine exchange (Figure 3E, bottom). Since salicylaldehyde 2f can displace the aliphatic chain of iminophospholipid 3a, it should be possible to permeabilize or disassemble aliphatic imine-constituted liposomes by adding a short-chain salicylaldehyde that can replace the long aliphatic tail, thus yielding an amphiphile that is unable to form membranes. We hypothesized that remodeling could be used to trigger the release of cargo encapsulated within the liposomes. We therefore studied the encapsulation and release of fluorophores using iminophospholipid liposomes (Figure 4). A solution of 1 in HEPES buffer was added to a mixture of a Cy5 dye and 2a in HEPES buffered solution (Figure 4B). After stirring the reaction for 16 h and removing the non-encapsulated fluorophore, we observed the presence of dye-loaded vesicles (Figure 4C). The addition of salicylaldehyde 2f and tris(2-carboxyethylphosphine) hydrochloride (TCEP·HCl), a known fluorescence quencher of Cy5 derivatives,33 followed by 10 h of stirring resulted in the disappearance of fluorescence along with the formation of new oil droplets (Figure 4D).

Figure 3. Reactivity of different aldehydes towards iminophospholipid formation and evidence for the dynamic nature of the system. A) Schematic representation of the dynamic imine formation and subsequent reduction. B) Structures of the aldehydes used herein. C) Relative amount of iminophospholipids after combining different aldehydes with 1 [measured after reduction of the mixture with NaBH4 and normalized using 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) as internal standard]. D) Ratio of formation of iminophospholipids 3a and 3d as a function of the temperature (measured as the relative yield of 4d versus 4a after reduction of the mixture). E) HPLC/ELSD traces after the addition of one equivalent of 2e (top) and 2f (bottom) to a 1 mM solution of 3a in HEPES solution, followed by reduction. Once the dynamic nature of the system and its capacity to distinguish between aldehydes had been proven, we decided to test whether addition of salicylaldehyde derivatives could induce the remodeling of one of the tails of the preformed sphingomyelin analog 3a (Figure 3E). As previously shown, salicylaldehyde 2e forms a very stable imine bond with 1, so we envisioned that 2e should be able to take the place of the oleylaldehyde 2a, affording sphingolipid 3e. As we expected, the addition of 2e to a solution of 3a in HEPES, followed by chemical reduction, led to the formation of 4e (Figure 3E, top). Exchanging a lipid tail of a phospholipid bears close resem-

Figure 4. Encapsulation and release of a fluorescent dye. A) Schematic representation showing the vesicle assembly from precursors 1 (dark blue) and 2a (light blue) and concurrent encapsulation of a Cy5 dye (green) followed by remodeling and quenching of the fluorophore. B) Fluorescence microscopy image of 1 and 2a in the presence of free dye immediately after mixing. C) Fluorescence microscopy image of iminophospholipid 3a vesicles containing the encapsulated fluorophore, after dialysis. D) Fluorescence microscopy image of the release and quenching of the Cy5 dye after treatment of vesicles from panel C with 2f (orange) and quencher TCEP·HCl (red). Insets represent images captured using phase-contrast microscopy, showing oil droplets before vesicle formation (B) and after the remodeling with 2f (D), while vesicles are present after imine formation (C). Scale bars denote 5 µm. In contrast, the addition of TCEP·HCl alone to a solution of oleylimine-3a-based vesicles containing the Cy5 dye resulted in negligible quenching of the encapsulated dye (Figure S7), demonstrating that the phosphine is unable to reach the confined Cy5 derivative unless membrane integrity is compromised by tail exchange. These experiments show how dynamic iminophospholipid vesicles

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can be modified to undergo drastic changes in the aggregation state of the supramolecular assemblies in response to a small molecule trigger. In summary, we have shown imine-based phospholipid analogs are capable of spontaneously self-assembling into bilayers. We have also established the relationship between the ability of the resulting iminophospholipids to form vesicles and the stability of a given imine bond. This approach constitutes the first example of dynamic combinatorial chemistry in phospholipid membranes, which mimics the Land’s cycle mechanism for membrane remodeling. Finally, iminophospholipid liposomes can respond to external stimuli, such as temperature and the addition of chemicals, which allows the release of cargo encapsulated within. We foresee future applications of dynamic phospholipid vesicles on the study of transient lipid membrane protein interactions, the development of vesicle carriers, and the depletion of lipids in proteoliposomes for the crystallization of membrane proteins.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed procedures, spectral data, Scheme S1, Tables S1-S2 and Figures S1-S7 (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected]

ORCID Neal K. Devaraj: 0000-0002-8033-9973 Andrés Seoane: 0000-0001-8908-4092 Roberto J. Brea: 0000-0002-0321-0156

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This material is based upon work supported by the Army Research Office through the MURI program under award number W911NF13-1-0383 and the National Science Foundation under CHE1254611. Andrés Seoane acknowledges Xunta de Galicia for his postdoctoral fellowship. Roberto J. Brea thanks the Human Frontier Science Program (HFSP) for his Cross-Disciplinary Fellowship. Alberto Fuertes acknowledges the Spanish Ministry of Education, Science and Sports (MECD) for his FPU Contract. TEM work was performed at the UC Irvine Materials Research Institute (IMRI). We acknowledge the use of the UCSD Cryo-Electron Microscopy Facility, which is supported by NIH grants to Dr. Timothy S. Baker and a gift from the Agouron Institute to UCSD.

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